This invention relates to background reduction devices and methods and more particularly to background reduction in confocal fluorescence detection systems.
Biological molecules are often tagged with fluorescent marker molecules for identification in analytical instruments in which the biological molecules are supported on a surface. The fluorescent signals originating from the surface (e.g., a glass substrate with fluorescently labeled molecules bound to it) can be weak so that the detection limit is often set by background radiation that comes from sources that lie outside of an intended detection plane. The contribution of such sources to the detected signal can be reduced substantially by using a confocal system as described extensively in the literature. See, for example, Handbook of Biological Confocal Microscopy, James Pawley, Ed. As is well known, confocal systems as used in scanners and microscopes reduce signal from out-of-plane sources. However, confocal systems have a normalized detection probability that goes to zero only asymptotically as a function of increasing distance from the intended plane of detection.
As an example, in the case of scanning a chip in a cartridge such as the Affymetrix GeneChip, non-negligible signals can be caused by fluorescent deposits on the front of the glass of the chip as well as by the illuminating laser beam hitting the back wall of the liquid cell in the chip cartridge. Confocality alone may not provide sufficient attenuation of such unwanted signals. One approach to reducing out-of-plane signals (such as from a back wall) when the confocal depth discrimination is insufficient is off-axis detection as used in a Hewlett-Packard G2500A system. However, off-axis detection is difficult to modify for large numerical aperture, i.e., high light collection efficiency. The present invention aims at specifically reducing unwanted background contributions for which confocality alone may not provide adequate suppression.